PROJECT SUMMARY/ABSTRACT Astrocytes perform essential homeostatic functions in the brain. In response to local tissue injury, astrocytes become ?reactive?, a process classically characterized by morphological hypertrophy and upregulation of GFAP. Reactive astrocytes are a defining feature of Alzheimer's disease (AD) neuropathology and are strongly correlated with cognitive decline in AD. However, mechanisms controlling astrocyte reactivity are not well understood. Recent studies have identified a form of reactive astrocyte induced by inflammatory activation (?inflammatory reactivity?, a.k.a. ?A1? reactivity) that may play a role in AD. Inflammatory reactive astrocytes are characterized by loss of normal homeostatic functions such as phagocytosis of CNS substrates as well as gain of neurotoxic properties. Importantly, they are found in a mouse model of AD as well as normal aged mice. Furthermore, human post-mortem AD brains are enriched for astrocytes expressing a marker of inflammatory reactivity. To elucidate mechanisms controlling inflammatory reactivity and its functional outputs in human astrocytes, in my preliminary work I implemented pooled CRISPRi loss-of-function screening in human iPSC- derived astrocytes (iAstros). From a preliminary screen, I identified myosin phosphatase as a potential regulator of the decreased phagocytic activity in inflammatory astrocytes. Furthermore, I leveraged existing astrocyte RNA-seq datasets to infer transcriptional regulators of inflammatory reactivity. In my first aim, I propose to determine how regulation of myosin phosphatase leads to decreased phagocytosis in inflammatory reactive astrocytes. In my second aim, I propose integrate genome-wide CRISPRi screening and coexpression analysis of existing astrocyte RNA-seq datasets to discover cellular pathways controlling inflammatory reactivity, followed by connecting these pathways to functional outputs of astrocyte reactivity. My sponsor Dr. Martin Kampmann, who co-developed the CRISPRi screening technology, and my co-sponsor Dr. Sergio Baranzini, an expert in neuroinflammation and the integration and analysis of transcriptomic datasets, are ideally positioned to support my proposed research. Furthermore, my collaborator Dr. Erik Ullian is an expert on the differentiation of high- quality astrocytes from human iPSCs, which further supports the feasibility of the proposed work. In addition to my two sponsors and my collaborator Dr. Ullian, I will also receive mentorship from Dr. Aimee Kao, a physician- scientist and practicing neurologist with whom I will undergo longitudinal clinical training in neurology, and Dr. Bruce Conklin, a global leader in iPSC-based technologies and genome surgery. Overall, the work proposed in this fellowship should contribute towards the development of drugs that can selectively modulate different functional outputs of astrocyte reactivity for the treatment of AD. Furthermore, through this work I will develop expertise in uncovering disease mechanisms with iPSC-derived models and functional genomics which will strengthen my training as a physician-scientist.